Transgenic Research 8: 265–277, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
265
Conditional gene targeting in macrophages and granulocytes using
LysMcre mice
B.E. Clausen1,4 , C. Burkhardt1 , W. Reith2 , R. Renkawitz3 & I. Förster1,5,∗
1 Institute
for Genetics, University of Cologne, Weyertal 121, 50931 Cologne, Germany; 2 Department of Genetics
and Microbiology, University of Geneva Medical School, CMU, 9 Ave. de Champel, 1211 Geneva 4, Switzerland;
3 Genetisches Institut, Justus-Liebig Universität, Heinrich-Buff-Ring 58–62, 35392 Giessen, Germany; 4 Present
address: Laborabory of Cellular Physiology and Immunology, The Rockefeller University, 1230 York Avenue, New
York, NY 10021, USA; 5 Present address: Institute for Medical Microbiology, Immunology and Hygiene,Technical,
University of Munich, Trogerstr 9, 81675 Munich, Germany
Received 25 January 1999; accepted: 15 April 1999
Key words: Cre-recombinase, macrophages, M-lysozyme, MHC class II, RFX5, gene targeting
Abstract
Conditional mutagenesis in mice has recently been made possible through the combination of gene targeting
techniques and site-directed mutagenesis, using the bacteriophage P1-derived Cre/loxP recombination system. The
versatility of this approach depends on the availability of mouse mutants in which the recombinase Cre is expressed
in the appropriate cell lineages or tissues. Here we report the generation of mice that express Cre in myeloid
cells due to targeted insertion of the cre cDNA into their endogenous M lysozyme locus. In double mutant mice
harboring both the LysMcre allele and one of two different loxP-flanked target genes tested, a deletion efficiency
of 83–98% was determined in mature macrophages and near 100% in granulocytes. Partial deletion (16%) could
be detected in CD11c+ splenic dendritic cells which are closely related to the monocyte/macrophage lineage. In
contrast, no significant deletion was observed in tail DNA or purified T and B cells. Taken together, LysMcre mice
allow for both specific and highly efficient Cre-mediated deletion of loxP-flanked target genes in myeloid cells.
Introduction
Cells of the myeloid lineage play a major role in
the maintenance of tissue homeostasis and immunological defense. In particular, the mononuclear phagocytes, including blood monocytes and resident tissue macrophages, have been demonstrated to be of
key importance for the establishment of innate immunity as well as the cytokine-mediated regulation of
acquired immune responses (Medzhitov & Janeway,
1998; Unanue & Allen, 1987). At the genomic level,
the differentiation of myeloid cells from multipotent
precursors is directed through the controlled expression of a series of myeloid-restricted genes. To date,
more than 30 genes specifically expressed in macro∗ Author for correspondence (Fax: +49 89 4140 7461; E-mail:
[email protected])
phages and granulocytes have been cloned and characterized, as recently reviewed by Clarke & Gordon
(1998).
To further explore differentiation and function of
myeloid cells in vivo it will be advantageous to
apply the Cre/loxP recombination system (for review, see Kühn & Schwenk, 1997; Rajewsky et al.,
1996) to conditional gene targeting in macrophages
and granulocytes. The bacteriophage P1-derived Crerecombinase catalyses sequence-specific DNA recombination between 34 bp spanning ‘loxP’ sites (Sternberg & Hamilton, 1981). To achieve conditional
Cre/loxP-mediated recombination in mice, loxP sites
have to be first introduced into the mouse genome
at the desired position by conventional gene targeting (Capecchi, 1989; Thomas & Capecchi, 1987).
Simultaneous cell type-specific expression of the Cre
recombinase in vivo then allows for deletion or in-
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version of the loxP -flanked (‘floxed’) target gene,
depending on the orientation of the loxP sites (Sternberg & Hamilton, 1981). In recent years an increasing
number of transgenic or gene-targeted mouse strains
have been established which express Cre in different cell types or tissues (for review, see Kühn &
Schwenk, 1997; Porter, 1998). Here, we are the first
to describe a mouse line which specifically expresses
Cre under control of the murine M lysozyme gene
in monocytes/macrophages and neutrophils (LysMcre
mice).
Both the human lysozyme promoter (Clarke et
al., 1996) and a genomic fragment covering the entire chicken lysozyme gene (Bonifer et al., 1994,
1990) have been successfully used to direct transgene
expression to myeloid cells in mice. Nevertheless, random insertion of transgenes in the mouse genome may
lead to variability of transgene expression depending
on the integration site and/or copy number. As an alternative to the conventional transgenic approach, and
to avoid species-specific differences in transcriptional
control (Bonifer et al., 1994), we decided to insert
the cre cDNA into the endogenous mouse M lysozyme gene precisely at its translational start site by
gene targeting. A similar ‘knock-in’ approach has previously been employed for generation of CD19-Cre
mice expressing Cre specifically in B cells (Rickert
et al., 1997). However, in the case of the LysMcre
mice, the neomycin resistance (neor ) gene required for
selection of targeted embryonic stem (ES) cells was removed from the targeted locus using the yeast-derived
FLP/FRT system (Broach & Hicks, 1980; Dymecki,
1996; O’Gorman et al., 1991). Thus, except for the insertion of cre and one remaining FRT site, the genomic
organization of the endogenous M lysozyme gene was
not altered.
In contrast to man, who has one lysozyme gene
(Peters et al., 1989), the murine genome encodes for
two lysozyme genes, the M gene specific for myeloid
cells and the P gene expressed in Paneth cells (Cross
& Renkawitz, 1990). The two murine lysozyme genes
were generated by a recent gene duplication event,
thus sharing a high degree of homology (Cortopassi &
Wilson, 1990; Cross & Renkawitz, 1990). Cell typespecific demethylation of a 30 enhancer downstream of
the mouse M lysozyme gene is involved in its myeloid
specific expression (Klages et al., 1992). In addition to
its constitutive expression (Bonifer et al., 1994; Cross
et al., 1988), up-regulation of lysozyme transcription
is an inducible marker of macrophage activation in
murine tissues (Keshav et al., 1991).
The efficiency of Cre-mediated deletion in LysMcre mice was directly assessed by crossing these
mice to two different mouse strains carrying loxPflanked target genes in their genome. First, we used
βT14 mice (Gu et al., 1994) which harbor a loxPflanked β polymerase (βpol) gene and have previously
been employed as an indicator of Cre-mediated deletion efficiency in vivo (Gu et al., 1994; Kühn et
al., 1995; Rickert et al., 1997). Second, LysMcre
mice were crossed to RFX5flox mice (Clausen et al.,
1998, and below). The transcription factor RFX5 is essential for both constitutive and Interferon-γ (IFNγ )inducible MHC class II (MHC-II) expression in macrophages (Clausen et al., 1998). Therefore, conditional
deletion of the RFX5 gene in macrophages should result in the loss of MHC-II expression. In this study,
we report on the efficiency of Cre-mediated deletion
of the floxed target genes in LysMcre/βpolflox and
LysMcre/RFX5flox mice as an initial characterization
of the LysMcre line.
Materials and methods
Mice
All experiments were performed with mice 8 to
13 weeks old. LysMcre, RFX5flox/flox, LysMcre/
RFX5flox/flox,
LysMcre/RFX5+/flox,
LysMcre
−/flox
and RFX5+/− mice, as well as βT14
RFX5
mice, were on a mixed Sv129/C57B1/6/CB.20 background, while Aα−/− mice were C57B1/6. Aα−/−
mice (Kontgen et al., 1993) were kindly provided by
H. Bluethmann (Basel, Switzerland). βT14 mice (Gu
et al., 1994) were from this laboratory. Aα−/− mice
were housed under SPF conditions while all other
animals were kept in a conventional animal facility.
Generation of LysMcre mice
Preparation of the LysMcre targeting construct was
performed by standard recombinant DNA technology
(Sambrook et al., 1989) starting from three consecutive genomic subclones derived from cosmid λg11
(Cross & Renkawitz, 1990) covering the entire M
lysozyme locus: 11B10 carried a 1.2 kb BamHI fragment immediate 50 of the M lysozyme gene, clone
11B5 harbored a 3.8 kb BamHI fragment covering the
promoter region and exons I−III, and 11B4 contained
a 7.5 kb BamHI fragment including exon IV of the M
lysozyme gene and its downstream region. The creNLS
cDNA was introduced into the endogenous ATG start
267
site of the M lysozyme gene by the following PCR
strategy: a 550 bp fragment was amplified from clone
11B5 using a 50 primer with a ClaI restriction site (BJ2: 50 - NNNATCGATACCATGGCAAGGAGTAAGG30 , and a 30 primer with a creNLS -specific overhang downstream of the ATG start (BJ-3: 50 TCTTCTTGGGCATGGTGACTGGAGGCTG-30).
Second, a 350 bp creNLS fragment was amplified using a 50 primer with a M-lysozymespecific overhang upstream of its ATG site (BJ4: 50 -TCCAGTCACCATGCCCAAGAAGAAGAGG30 ), and a 30 primer around the unique BamHI site
present in the cre cDNA (BJ-5: 50 -NNNNGGATCCGCCGCATAACCAGTGAA-30 ). Next, both fragments were included in a PCR reaction together
with the two outer primers (BJ-2 and BJ-5) to
generate the 1 kb LysM/creNLS ‘bridge’ fragment.
The PCR product was treated with ClaI and
BamHI for trimolecular ligation with the downstream BamHI/XbaI creNLS -fragment (derived from
pEµNLScrehGH, gift of F. Schwenk) and a ClaI/XbaI
opened pBSIIKS (pSAHCre2). Clone 11B5 was then
used to generate the downstream long arm of homology (LAH) of the targeting vector: the entire genomic
BamHI-fragment of clone 11B5 was partially digested
with BbsI to recover the 3.25 kb LAH. 50 overhangs
were filled in with Klenow followed by blunt-end ligation into EcoRV-digested pUC19-DL (pLAH23). To
prolong the SAH to 1.3 kb, an upstream fragment
was amplified from genomic DNA (11B10) using a
50 oligo containing a XhoI restriction site (BJ-16: 50 NNNNCTCGAGAACACCATGCTCGGCTAG-30 ) in
combination with a 30 oligo complementary to
the region around a unique NsiI site present in
the SAH derived from clone 11B5 (BJ-14: 50 TGGAATGCATTTATCCTTTTTC-30). This fragment
was then introduced into XhoI/NsiI-digested pSAHCre2 (pSAH+CreReal3). The strategy to prolong the
LAH was to recover the 50 3.3 kb BamHI/XbaI fragment of genomic clone 11B4, remove 50 protruding
ends with Klenow, and to insert this piece into SacII
opened and blunt-ended pLAH23 (pLAH+4). The
construct was then finalized by subsequent introduction into the 30 SalI site of pSAH+CreReal3 of (1)
a XhoI/SalI-cut tk gene (1.9 kb tk gene derived from
pIC19R-MC1tk); (2) XhoI/SalI-cut FRT-flanked neor
(1.5 kb FRT2neo cassette recovered from pFRT2neoA
(Jung et al., 1993); and (3) the extended LAH (cloned
as a SalI fragment from pLAH+4). Integrity of the
Cre cDNA was verified by sequence analysis (data
not shown). The FRT sites flanking the neo selection
marker were functionally tested in vitro by ‘digestion’
of the final targeting vector with a FLP- containing
crude protein extract (gift from J. Roes), and the excised neor circle was visualized on a 0.6% agarose
gel after FLP-mediated deletion (1.3 kb, supercoiled)
(data not shown). Although functional, the FRT sites
in pFRT2neoA were found to contain a point mutation in the additional 13 bp repeat as compared to the
published sequence (Senecoff et al., 1985; L. Pao,
unpublished data).
In parallel, a test-construct was generated to establish PCR conditions to facilitate screening for homologous recombinants in tES cells (see below). Primer
pair BJ-15 (50 -NNNNCTCGAGCAGCCTATTATCTGAAGG- 30 )/BJ-14 was used to amplify and clone
a slightly longer SAH of 1.5 kb from genomic DNA
into pSAHCre2 (pSAH+CreTest3). To complete the
test-construct, the neor selection marker (XhoI/SalIfragment from pFRT2neoA) was inserted into the 30
SalI site. The PCR was performed using an oligo
50 of the SAH (BJ-15) and a Cre-specific primer
(BJ-17:50 -CGGTCAGTAAATTGGACAC-30).
The targeting construct was linearized with XhoI
50 of the SAH and transfected into E14.1 ES cells by
electroporation. Homologous recombination in PCRpositve G418/gancyclovir double-resistant colonies
was verified by genomic Southern blot analysis using an EcoRI digest in combination with an external
probe outside the SAH (probe A). To remove the
FRT-flanked neor marker two independent clones,
tLysMcre126 and tLysMcre176, were expanded and
subjected to FLP-mediated neo deletion by transient
transfection with the FLP expression vector pOG44fix
(gift from S. O’Gorman). Clone tLysMcre1261neo97
was identified by Southern blot analysis as carrying the neo-deleted targeted M lysozyme allele and
was injected into blastocysts to generate mutant LysMcre mice according to standard procedures (Torres
& Kühn, 1997).
LysMcre mice are routinely typed by PCR using
the primer pair NLSCre (50 -CCCAAGAAGAAGAGGAAGGTGTCC-30 ) and Cre8 (50 -CCCAGAAATGCCAGATTACG-30 ).
RFX5floxmice
ES cell clones carrying a ‘floxed’ RFX5 allele were
isolated by deleting the neor gene from the original
targeted RFX5 locus (Clausen et al., 1998) by transient Cre transfection, and germline transmission of
the floxed RFX5 allele was obtained after microin-
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jection of the mutant ES cell clones into CB.20
blastocysts (B.E.C., W.R. and I.F., data not shown).
The mice are routinely typed by PCR, using the
same primer combination as for typing of RFX5+/−
mice (N4, N8, INT1) (Clausen et al., 1998). The
‘floxed’ RFX5 allele yields a ca. 300 bp fragment
that can be distinguished from the wild type (ca.
250 bp) and deleted (ca. 500 bp) allele by agarose gel
electrophoresis.
Cell preparation and BM cultures
LN T cells and splenic B cells were positively enriched
from single cell suspensions by magnetic cell sorting
(MACS, Miltenyi Biotec, Germany) using anti-Thy1.2
and anti-B220 MicroBeads, respectively. Resident
peritoneal macrophages were harvested by washing
the peritoneal cavity with 10 ml of medium. Likewise, neutrophilic granulocytes were obtained from
the peritoneal cavity following i.p. injection of 1–
2 ml of thioglycollate 4 h prior to cell harvest (Liu et
al., 1996). Splenic dendritic cells (DC) were prepared
after collagenase digest of the organ as described
(Crowley et al., 1989) (collagenase D, Boehringer
Mannheim). To facilitate FACS sorting, the DC were
positively MACS- enriched from total splenocytes
using anti-CD11c MicroBeads.
DC and macrophages were generated from BM
precursors according to a protocol modified from
Inaba et al. (1992). Briefly, BM cell suspensions
were depleted of mature T cells (anti-Thy1.2 MicroBeads), B cells (anti B220 MicroBeads) and MHCII+ cells (biotin-conjugated M5/114 in concert with
SA-coupled MicroBeads) by MACS. The remaining BM precursors were cultured for 7 days in the
presence of low amounts of GM-CSF (10% of culture supernatant of X63 hybridoma transfected with a
GM-CSF expression vector (Karasuyama & Melchers,
1988) to drive differentiation along the DC lineage.
DC were sorted as CD11c+ /MHC-II+ /Gr1− /B220−
cells. Differentiation into macrophages was achieved
by culture of lineage-depleted BM precursors with MCSF (30% of L cell-conditioned medium (Stanley &
Heard, 1977; Tushinski et al., 1982). For in vitro stimulation the BM-derived macrophages were cultured
for the last day with 400 U/ml of recombinant mouse
IFNγ (Genzyme Diagnostics, USA). Macrophages
were sorted as F4/80+ /MHC-II+ /B220− cells.
Cytofluorometric analysis
Fluorescence staining was performed as previously
described (Forster & Rajewsky, 1987). Cells were
analysed on a FACScan (Becton Dickinson) or sorted on a FACStar (Becton Dickinson). The following antibodies were used in the experiments: Ly-6G
for Gr1 and HL3 for CD11c were purchased from
Pharmingen. Macrophages were identified by anti
F4/80 (MCA497F, Serotec, UK) staining. RA3-6B2
for B220 (Coffman, 1982); and M5/114 for MHCII (Bhattacharya et al., 1981) were purified from
hybridoma supernatants in our laboratory.
Genomic Southern blot analysis
Southern blot analysis was performed by standard procedures (Sambrook et al., 1989) For detection of cre
the 1.45 kb SalI fragment from pEµNLScrehGH was
used as a probe, while the neor cassette was detected
by probing with the 1.5 kb SalI/XhoI fragment from
pFRT2neoA. Analysis of the βpol gene was performed
using the 0.76 kb HindIII/BamHI fragment derived
from pMGβ-2 (Gu et al., 1994). Other probes used
in the course of generating LysMcre and RFX5flox
mice are described in Figures 1 and 3. The 50 MRFX5
probe was the same as described in (Clausen et al.,
1998); the 410 bp 30 MRFX5 probe was isolated from
genomic DNA by PCR amplification using primers
RFX5-7(50 - GTGTGGATGGACAGGTGTGC-30) and
RFX5-8 (50 -GGGTCACTGCAGGAGGGTCC-30 ). The
PCR product was cloned into the pGEM-T vector (Promega) and isolated by SacII/SpeI digestion. Radioactive blots were exposed to a PhosphoImagerscreen (Fuji) and analysed on a Bio-ImagingAnalyzer (Fuji Bas 1000).
Results
Generation of LysMcre-expressing mice
The targeting vector was constructed such that the
cDNA for Cre was introduced into the endogenous
ATG-start site within the first exon of the M lysozyme gene (Figure 1A). To facilitate translocation
into the nucleus, cre was genetically engineered to
encode a nuclear localization signal (NLS) (Gu et
al., 1993). With the intention not to interfere with
the M lysozyme-specific 30 enhancer (Klages et al.,
1992) nor to disturb the function of potential unknown elements driving cell type-specific expression
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Figure 1. Targeting strategy for introduction of cre into the M lysozyme gene. A. Top: Genomic organization of the two lysozyme genes in the
mouse. Middle: Targeting vector containing the creNLS cDNA (Gu et al., 1993) at the endogenous ATG start site, followed by a FRT-flanked
neor cassette (Jung et al., 1993). The HSV-tk gene was cloned downstream of the LAH to select against random integrants. Bottom: Targeted
allele after homologous recombination in ES cells. Probes used for Southern blot analysis are shown as black bars: A, ext. SAH; B, ext.
LAH; C, cre probe. Vertical arrows indicate the borders of the targeting vector; horizontal arrows indicate PCR primers used for screening of
G418/gancyclovir double-resistant colonies. SAH, short arm of homology (1.4 kb); LAH, long arm of homology (6.6 kb); Ba, BamHI; E, EcoRI.
B. Southern blot showing homologous recombination in ES cells (probe A). wt, wild-type E14.1 ES cells; 125, 126, and 175, 176 represent
pools of two ES cell colonies that were positive in the initial PCR screen. Out of those, clone 126 and 176 were identified as homologous
recombinants (tLysMcre 126 and 176, respectively). ? = cross-hybridization of the Southern probe to a band of unknown identity. C. Southern
strategy to verify FLP-mediated neor deletion of tLysMcre homologous recombinants. Targeted ES cell clones before (top) and after (bottom)
neo deletion. Sizes of expected restriction fragments are indicated. C, internal cre probe; Ba, BamHI; Bg, BglII, As indicated, one of the BamHI
sites was destroyed during cloning of the targeting vector but this mutation was not cointegrated into the targeted allele of clone 126. D. Southern
blots of FLP-mediated neor deletion in vitro (probe C). tLysMcre126, 176, targeted parental ES cell clones; 1neo126, neo-deleted subclone
97; wt, wild-type E14.1 ES cells. E. Germline transmission of the LysMcre mutation (probe C.) +, tES cell clone tLysMcre1261neo97; #5 and
#6 are two germline mice as scored by coat colour, #5 being one of the founders of the LysMcre line.
270
Figure 1. (Continued).
of M lysozyme, the downstream organization of the
gene was left untouched. Therefore, stabilization of
the transcript should be achieved by normal splicing
within the M lysozyme gene and utilization of the
endogenous polyadenylation site. To select for homologous recombinants, a FRT-flanked neor gene (Jung
et al., 1993) was cloned immediately downstream
of the creNLS cDNA, and a herpes simplex virusthymidin kinase (HSV-tk) gene was inserted 30 of the
long arm of homology (LAH) (Figure 1A). The linearized targeting construct was transfected into E14.1
ES cells by electroporation, and G418/gancyclovir
double-resistant ES cell colonies were pre-screened
by PCR (data not shown). Homologous recombination
was then verified by genomic Southern blot analysis
using an EcoRI digest in combination with an external
probe outside of the short arm of homology (SAH)
(Figure 1A and B, probe A, and data not shown). The
targeting frequency was 1 out of 15 double-resistant
colonies.
As a result of a recent gene duplication event, the
two murine lysozyme genes are located in close tandem repeat, only about 5 kb apart from each other
(Figure 1A). Both genes share an identical exon/intron
organization and are highly homologous (Cross &
Renkawitz, 1990). Nevertheless, none of the targeted
clones showed a restriction pattern indicative of a recombination event within the P lysozyme gene (data
not shown).
To remove the FRT-flanked neor selection marker,
two independent clones, tLysMcre126 and tLysMcre176, were expanded and subjected to FLPmediated neor deletion. Southern blot analysis of
G418-sensitive clones identified neor -deleted ES cell
clones with a frequency of 3 out of 400. As evident
from Figure 1C and D, clone tLysMcre1261neo97
carried the neor -deleted targeted M lysozyme allele.
This clone was injected into blastocysts to generate
LysMcre mutant mice (Figure 1E).
Efficient and cell type-specific Cre-mediated deletion
of a loxP-flanked β polymerase gene
To assess cell type specificity and efficiency of Cremediated deletion, LysMcre mice were first crossed
with βT14 mice (Gu et al., 1994) which are homozygous for a loxP-flanked βpol gene. Genomic
organization of the wild-type and targeted locus of
the βpol gene, as well as the corresponding restriction pattern after BamHI digestion of genomic DNA,
are illustrated in Figure 2C. Deletion of the target
gene was quantified by genomic Southern blot analysis of LysMcre/βpol+/flox double-mutant mice (Figure 2). Deletion was nearly complete in FACS-purified
F4/80+ peritoneal macrophages (95%, Figure 2A and
D), and in thioglycollate-elicited, sorted Gr1+ peritoneal neutrophils (99%, Figure 2A and D). The faint
band indicating Cre-mediated deletion in genomic
DNA preparations of total lung and spleen can be attributed to resident macrophage populations present
in these tissues (Figure 2A). MACS-enriched lymph
node (LN) T cells and splenic B cells as well as
tail DNA did not show significant deletion of the
loxP-flanked βpol target gene (Figure 2A and D).
However, about 16% deletion was detected in sorted
CD11c+ dendritic cells (DC) (Figure 2A and D, and
see discussion).
To determine the deletion efficiency during early
in vitro differentiation of the relevant cell populations,
BM cells were cultured in the presence of M-CSF (for
BM-derived macrophages) (Stanley & Heard, 1977;
Tushinski et al., 1982), or GM-CSF (for BM-derived
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Figure 2. Southern blot analysis of Cre-mediated deletion in LysMcre/βpol+/flox double transgenic mice. A. Cell type-specific deletion in ex
vivo cell preparations. B. Deletion efficiency in in vitro differentiated cell populations. C. Strategy to distinguish the βpol wt (top), loxP-flanked
(middle) and deleted (bottom) alleles which are represented by BamHI fragments of 10, 4.5 and 3 kb, respectively. The probe used for Southern
hybridization is indicated as a black bar. D. Quantification of the deletion efficiency in selected lanes of the Southern blots shown in A and B.
Mφ, macrophages; DC, dendritic cells; Sp, spleen; B, BamHI.
DC and granulocytes) (Inaba et al., 1992; Scheicher
et al., 1992), respectively. Deletion of the βpolflox
allele was less efficient in BM-macrophages and BMgranulocytes (Figure 2B and D, 75% and 79%, respectively) compared to mature cells isolated from the
peritoneal cavity. In contrast, there was a higher degree of deletion of the floxed βpol gene in BM-derived
DC than in splenic CD11c+ DC (Figure 2B and D,
31%).
Efficient deletion of a loxP-flanked RFX5 target gene
in mature macrophages
To verify the high efficiency of macrophage-specific
Cre-mediated recombination in LysMcre mice for a
second loxP-flanked gene segment, the animals were
crossed to a strain harboring a RFX5flox mutation in
the germline. RFX5flox mice were generated from
the RFX5-targeted ES cell clone #50 (Clausen et al.,
1998) after Cre-mediated deletion of the neor marker
(see also Materials and Methods). The RFX5flox allele is characterized by two loxP sites flanking the
exons encoding the DNA binding domain (DBD) of
the transcription factor (see Figure 3A). In RFX5flox
mice, surface MHC-II expression was normal as assessed by FACS analysis of B220+ splenic B cells
(data not shown), indicating that the intronic loxP sites
are placed such that they do not interfere with the expression of the RFX5 protein. In conditional mouse
mutants this is critically important to ensure normal
expression of the floxed allele in those cell types that
are not targets of Cre-mediated deletion.
Efficiency of Cre-mediated deletion of the loxPflanked RFX5 allele was initially determined by genomic Southern blot analysis of FACS-purified F4/80+
macrophages from the peritoneal cavity of a group
of 9 LysMcre/RFX5flox/+ mice (Figure 3). Controls
include total peritoneal cells and spleen cells from
272
Figure 3. Deletion efficiency of a loxP-flanked RFX5 target gene in macrophages of LysMcre/RFX5flox/+ mutant mice. A. Genomic RFX5
locus (top), loxP-flanked RFX5 allele after partial neor deletion (middle) and deleted allele after conditional Cre-mediated deletion in vivo
(bottom) (see also (Clausen et al., 1998)). Restriction fragments obtained with the 50 MRFX5 probe after XbaI digest and with the 30 MRFX5
probe after EcoRI digest are indicated. DBD, DNA-binding domain; X, XbaI; R, EcoRI. B. Southern blot analysis of EcoRI-digested DNA
using the 30 MRFX5 probe. Left: DNA derived from total peritoneal exsudate cells, spleen cells, and FACS-purified F4/80+ macrophages
from a pool of 9 LysMcre/RFX5flox/+ mice (deletion efficiency for each sample is indicated); Right: control tail DNA of LysMcre/RFX5 +/+ ,
LysMcre/RFX5+/− and LysMcre/RFX5−/− mice.
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Figure 4. MHC-II surface expression correlates with genomic deletion of the RFX5flox gene. A. FACS analysis of MHC-II expression on
F4/80+ macrophages differentiated from BM-precursors in vitro. LysMcre/RFX5−/flox mice are compared with Aα−/− and RFX5+/−
controls. Histograms show MHC-II expression in the absence of IFNγ (top), and after IFNγ treatment for the last 24 h of culture (bottom).
The table summarizes the percentages of MHC-II+ macrophages generated in the same cultures. B. Southern blot analysis of deletion of the
RFX5flox allele in sorted MHC-II− and MHC- II+ macrophages from BM-cultures of LysMcre/RFX5−/flox mice as shown in A. DNA was
digested with XbaI and hybridized with the 50 MRFX5 probe (see Figure 3A). Numbers represent individual mice. pool, MHC-II+ cells pooled
from mice 1043, 1044 and 1092. The table below gives a quantification of the deletion efficiency.
the same mice, as well as tail DNA from RFX5+/+ ,
RFX5+/− , and RFX5−/− mice (Figure 3B). The genomic organization of the different RFX5 alleles and
the expected restriction pattern of an EcoRI digest in
concert with the 30 MRFX5 probe are shown in Figure 3A. As is evident from Figure 3B, the 3.2 kb band
indicative of the RFX5flox allele is only very faint in
purified macrophages, while a strong signal can be
detected in total peritoneal exsudate cells and spleen
cells. In this experiment, the deletion efficiency was
calculated to be 83.2% for the FACS-purified F4/80+
macrophages. In total peritoneal cells and spleen cells,
the proportion of the deleted RFX5 allele was 22.5%
and 2.5%, respectively, in good agreement with the
relative proportion of macrophages and granulocytes
in these cells populations. A similar deletion efficiency
was observed in an independent experiment using
LysMcre/RFX5flox/− mice (data not shown). These
findings demonstrate that deletion of the RFX5flox
allele in LysMcre mice occurs with high efficiency
though somewhat lower than that of the loxP-flanked
βpol target gene (see above).
Partial deletion of the RFX5flox allele and residual
MHC-II expression on BM-derived
LysMcre/RFX5−/flox macrophages
The transcription factor RFX5 is required for MHCII expression on murine macrophages (Clausen et
al., 1998). We therefore assessed whether cell typespecific Cre-mediated mutation of the RFX5 gene in
BM cells of LysMcre/RFX5flox mice results in the
loss of MHC-II surface expression. First, constitutive
surface MHC–II expression on BM-derived F4/80+
macrophages was analysed by FACS (Figure 4A, top).
BM-derived macrophages from conventional MHC-II-
274
deficient Aα−/− mice (Kontgen et al., 1993) were used
as negative controls. In contrast to these Aα−/− macrophages (Figure 4A, top, black line), a small fraction
of LysMcre/RFX5−/flox macrophages expressed surface MHC-II (6.2%, Figure 4A, top, shaded peak).
This subpopulation was significantly reduced compared to RFX5+/− macrophages which are characterized by wild type MHC-II levels (30%, Figure 4A, top,
gray line).
In a separate experiment, induction of surface
MHC-II expression was assessed by FACS-analysis
of BM-derived macrophages from Aα−/− , RFX5+/−
and LysMcre/RFX5−/flox mice upon stimulation with
IFNγ for the last 24 h of in vitro differentiation (Figure 4A, bottom). While background fluorescence of
Aα−/− macrophages did not increase significantly
after IFNγ stimulation (4.7%; Figure 4A, bottom,
black line), the majority of RFX5+/− macrophages
expressed MHC-II in response to IFNγ activation
(72%, Figure 4A, middle, gray line). In contrast, only
a minor fraction of LysMcre/RFX5−/flox macrophages
could be induced to express MHC-II (19%; Figure 4A,
middle, shaded peak) upon IFNγ treatment. Nevertheless, there appears to be an increase in the proportion
of surface MHC-II+ cells as compared to uninduced
cultures.
Deletion of RFX5 in LysMcre/RFX5−/flox BM-derived
macrophages correlates with MHC-II expression
In the light of the residual MHC-II expression in
macrophages derived from BM precursors of the
conditional mutants, it was of interest to determine
whether the absence or presence of surface MHC-II
in LysMcre/RFX5−/flox macrophages correlated with
the state of deletion of the loxP-flanked RFX5 allele.
Therefore, macrophages were differentiated in M-CSF
conditioned BM cultures with or without IFNγ activation during the last day of culture. The respective
FACS-sorted F4/80+ MHC-II− and F4/80+ MHC-II+
subpopulations were analyzed by Southern blotting
(Figure 4B) using Xba–digested DNA and the 50
MRFX5 probe as depicted in Figure 3A. Irrespective of the IFNγ treatment, there was almost complete
deletion of the RFX5flox allele in the MHC-II− fraction of macrophages (Figure 4B, 96–98% in untreated
F4/80+ macrophages, and 86–96% in IFNγ -activated
macrophages). On the other hand, only little deletion
was detected in MHC-II+ subpopulations. In the absence of IFNγ , 38% of F4/80+ MHC-II+ macrophages
carried a deletion of the loxP-flanked RFX5 allele,
while no more than 0–10% of MHC-II+ macrophages
harbored a deleted target gene after IFNγ stimulation.
In conclusion, Cre-mediated deletion of the
RFX5flox allele represents an ongoing process in BM
cultures of LysMcre/RFX5−/flox mice. However, once
deletion of RFX5 has occured at the genomic level,
MHC-II surface expression appears to be lost within a
short period of time.
Discussion
LysMcre mice are engineered such that the cre cDNA
is inserted into the translational start site of the endogenous M lysozyme gene, driving its specific expression in monocyte/macrophages and neutrophils
(Cross et al., 1988; Cross & Renkawitz, 1990). Introduction of a FRT-flanked neor cassette instead of
a loxP-flanked neor gene allowed to remove the neor
gene by Flp-mediated recombination without leaving
behind an unwanted additional loxP site. Thus, potentially deleterious inter-chromosomal rearrangements
in LysMcre double mutant mice harboring a loxPflanked target gene are avoided (Abremski et al., 1983;
Hamilton & Abremski, 1984). In addition, removal of
the neor marker should ensure that the transcriptional
control of the targeted LysMcre allele is very similar
if not identical to its wild-type counterpart.
A potential risk of the ‘knock-in’ approach is that
the physiological transcription levels of the respective gene locus may not be high enough to achieve
sufficient expression of the Cre enzyme for recombination to occur. Transcription of lysozyme starts early
during development, is low but detectable in resident
tissue macrophages, and can be substantially elevated
in response to infectious agents (Bonifer et al., 1994;
Cross et al., 1988; Keshav et al., 1991; Scheinecker
et al., 1995). Our finding that the deletion efficiency
of floxed target genes in developing macrophages of
M-CSF stimulated BM cultures was close to 80%,
demonstrates that the amount of Cre enzyme is in the
majority of these cells sufficient to mediate deletion.
Analysis of the deletion efficiency in resident tissue
macrophages from the peritoneal cavity or spleen, as
well as in peritoneal granulocytes, further indicates
that deletion of floxed target genes is more complete in
these fully differentiated cells (83–99%; Figure 2A,2D
and 3B). Deletion was also found to be highly cell
type-specific, as it was not detected in LN T cells and
splenic B cells (both 2%; Figure 2A and D). Faint
signals observed in genomic DNA prepared from total
275
organs, i.e. lung and spleen (Figure 2A and D), can be
attributed to resident macrophage populations present
in these tissues. Taken together, these data demonstrate that LysMcre-mediated deletion at the genomic
level seems to be specific and highly efficient for at
least two independent target genes.
DC belong to the group of so called professional antigen presenting cells in the immune system
(Banchereau & Steinman, 1998). Although DC themselves represent a heterogeneous population, a major
fraction is known to be derived from myeloid precursors in the BM (Inaba et al., 1992; Scheicher et
al., 1992) and differentiation of monocytes into DC
has been described in cell culture (Pickl et al., 1996;
Sallusto & Lanzavecchia, 1994). Therefore, it was of
interest to determine whether LysMcre-mediated deletion could also be detected in DC. Indeed, we observed
that 16% of sorted CD11c+ splenic DC (Figure 2A
and D) and 31% of GM-CSF stimulated BM-derived
DC (Figure 2B and D) deleted the floxed βpol allele.
The higher incidence of deletion seen in BM-derived
DC is particularly intriguing since GM-CSF simultaneously mediates the differentiation of both DC and
macrophages from mouse BM precursors (Inaba et al.,
1992; Scheicher et al., 1992). Thus, the partial deletion observed in these cultures could be explained by
the existence of a common M lysozyme+ precursor of
myeloid DC and macrophages. Alternatively, it is also
possible that the sorted CD11c+ cells contain some
contaminating macrophages, or that a distinct subpopulation (or developmental stage) of DC expresses
M lysozyme. A recent report describes the expression
of lysozyme by DC which were in vitro differentiated
from human CD14+ peripheral blood monocytes in
the presence of GM-CSF plus IL-4 (Pickl et al., 1996).
In light of these various possibilities, the nature of the
cell type which is responsible for the partial deletion
seen in CD11c+ DC preparations remains to be identified. For the purpose of conditional gene targeting
experiments using LysMcre mice, we would like to
point out however that the majority of peripheral DC
is not affected by Cre-mediated deletion.
RFX5−/− macrophages were previously shown to
be devoid of MHC-II irrespective of in vitro stimulation with IFNγ (Clausen et al., 1998). This
enabled us to distinguish MHC-II+ from MHCII− macrophage populations in BM cultures from
LysMcre/RFX5− /flox precursors (Figure 4A) and
to assess their respective degree of deletion of the
RFX5flox allele by genomic Southern blot (Figure 4B).
As is evident from Figure 4A, M-CSF-conditioned
BM cultures from LysMcre/RFX5−/flox mice contained 4–5 fold less surface MHC- II+ macrophages
than BM cultures from wild-type (RFX5+/− ) mice.
The residual MHC-II expression on a fraction of
developing macrophages can most likely be attributed to the presence of cells in these cultures that
did not yet successfully delete the RFX5flox allele.
In this respect, these findings corroborate the reduced deletion efficiency of 79% of a polβflox allele
detected in F4/80+ BM-derived macrophages from
LysMcre/polβ+/flox mice (Figure 2B and D; see also
above). Alternatively, dependent on their half life, surface MHC-II molecules may persist for some time on
the surface of the cells despite efficient deletion of
the RFX5flox target. Genomic Southern blot analysis
of sorted MHC-II+ and MHC-II− macrophage subpopulations in these cultures revealed that deletion in
the MHC-II− cells was nearly complete (Figure 4B,
average of 97% and 91%, respectively). In contrast, in
the MHC-II+ fractions deletion occured in only 38%
of the cells without, and about 5% of the macrophages
after IFNγ incubation. The finding of only a minority
of RFX5-deleted macrophages amongst the MHC-II+
population, speaks against a prolonged persistence
of MHC-II molecules on the surface of macrophages
after deletion of the RFX5 gene. A more detailed analysis of the biological effects of conditional MHC-II
ablation in macrophages of LysMcre/RFX5flox mice is
presently being assessed.
Clarke and Gordon have recently argued that despite the availability of a series of myeloid-specific
promoters, transgene expression can generally not be
achieved in all stages of development and in all subsets of myeloid cells (Clark & Gordon, 1998). Use of
the Cre/loxP recombination system might overcome at
least part of this problem, as even transient expression
of the Cre-recombinase during development leads to
irreversible recombination of the floxed target genes
in all progeny cells. In the experiments described
here, we have used LysMcre mice to permanently inactivate genes in the myeloid lineage. Alternatively,
similar strategies can also be employed for the inducible expression of genes, for example by deletion
of artificially inserted transcriptional stop sequences
(Lakso et al., 1992). Thus, LysMcre mice should prove
to be a valuable tool for conditional mutagenesis in
monocytes/macrophages and neutrophils.
276
Acknowledgements
We are grateful to K. Rajewsky for his continuous support; to A. Egert and C. Göttlinger for expert technical
help; and to H. Jacobs, I. Lieberam, F. Rieux-Laucat,
C. Schmedt, F. Schwenk and R. Torres for advice and
discussion. This work was supported by the Deutsche
Forschungsgemeinschaft through SFB 243 and the
Volkswagen Foundation.
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